Beruflich Dokumente
Kultur Dokumente
Review
Agricultural Lipid Biotechnology Program, Department of Agricultural, Food, and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6H 2P5
Department of Pediatrics, Group on Molecular and Cell Biology of Lipids, University of Alberta, Edmonton, Canada T6G 2S2
c
Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E5
b
a r t i c l e
i n f o
Article history:
Available online 15 June 2012
Keywords:
Neutral lipids
Storage lipids
Triglyceride
Kennedy pathway
Obesity
Lipid biotechnology
a b s t r a c t
Triacylglycerol (TG) is a storage lipid which serves as an energy reservoir and a source of signalling molecules and substrates for membrane biogenesis. TG is essential for many physiological processes and its
metabolism is widely conserved in nature. Acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20)
catalyzes the nal step in the sn-glycerol-3-phosphate pathway leading to TG. DGAT activity resides mainly
in two distinct membrane bound polypeptides, known as DGAT1 and DGAT2 which have been identied in
numerous organisms. In addition, a few other enzymes also hold DGAT activity, including the DGATrelated acyl-CoA:monoacylglycerol acyltransferases (MGAT). Progress on understanding structure/function in DGATs has been limited by the lack of detailed three-dimensional structural information due to
the hydrophobic properties of theses enzymes and difculties associated with purication. This review
examines several aspects of DGAT and MGAT genes and enzymes, including current knowledge on their
gene structure, expression pattern, biochemical properties, membrane topology, functional motifs and
subcellular localization. Recent progress in probing structural and functional aspects of DGAT1 and DGAT2,
using a combination of molecular and biochemical techniques, is emphasized. Biotechnological applications involving DGAT enzymes ranging from obesity therapeutics to oilseed engineering are also discussed.
2012 Elsevier Ltd. All rights reserved.
Contents
1.
2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.
TG biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.
Role of DGAT in TG bioassembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Identification and molecular characterization of DGATs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
Dgat1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
Dgat2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.
DGAT2-related enzymes in mammals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4.
WS/DGAT in bacteria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.5.
Soluble DGAT-related enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
351
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353
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354
354
354
Abbreviations: ACAT, acyl-CoA:cholesterol acyltransferase; ACBP, acyl-CoA binding protein; ARAT, acyl-CoA:retinol acyltransferase; ASO, antisense oligonucleotide;
AWAT, acyl-CoA:wax alcohol acyltransferase; BSA, bovine serum albumin; CE, cholesteryl esters; CPM, 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin; DCR,
defective cuticle ridge; DG, diacylglycerol; DGAT, acyl-CoA:diacylglycerol acyltransferase; DGTA, diacylglycerol transacylase; EMS, ethyl methanesulfonate; ER, endoplasmic
reticulum; FA, fatty acid; G3P, sn-glycerol-3-phosphate; GPAT, acyl-CoA:sn-glycerol-3-phosphate acyltransferase; HMM, Hidden Markov Model; HTS, high throughput
screening; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; LPAT, acyl-CoA:lysophosphatidic acid acyltransferases; MAM, mitochondria-associated membranes;
MFAT, multifunctional O-acyltransferase; MG, monoacylglycerol; MGAT, acyl-CoA:monoacylglycerol acyltransferase; NBD, N,N0 -dimethyl-N(acetyl)-N0 -(7-nitrobenz-2-oxa1,3-diazol-4-yl) ethylene diamine; NEM, N-ethylmaleimide; PA, phosphatidic acid; PAP, phosphatidic acid phosphohydrolases; PC, phosphatidylcholine; PDAT,
phospholipid:diacylglycerol acyltransferase; PKA, protein kinase A; QTL, quantitative trait loci; SCD1, stearoyl-CoA desaturase 1; TG, triacylglycerol; TMD, transmembrane
domain; WS, wax ester synthase.
Corresponding author. Address: University of Alberta, Department of Agricultural, Food and Nutritional Science, 4-10 Ag/For Centre, Edmonton, AB, Canada T6G 2P5. Tel.:
+1 780 492 4401; fax: +1 780 492 4265.
E-mail addresses: qin.liu@ualberta.ca (Q. Liu), rodrigo.siloto@ales.ualberta.ca (R.M.P. Siloto), richard.lehner@ualberta.ca (R. Lehner), scot.stone@usask.ca (S.J. Stone),
randall.weselake@ualberta.ca (R.J. Weselake).
1
Present address: Department of Biology, Brookhaven National Laboratory, Upton, NY 11973, USA.
0163-7827/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.plipres.2012.06.001
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1. Introduction
Triacylglycerol (TG) is a non-polar acyl triester of glycerol and
fatty acids (FAs) [1]. As a major class of neutral lipid, the presence
of TG is widespread in eukaryotes including animals, plants and
fungi. In prokaryotes, TG accumulation is also a common feature
in species belonging to the actinomycetes group [2,3]. TG has multiple functions in living organisms, but serves principally as a reservoir of FAs for energy production. The synthesis and mobilization
of TG are essential cellular processes to maintain lipid and energy
homeostasis in all types of cells [46]. In mammals, TG is important for energy storage in adipose tissue. Also, it protects cells from
lipotoxicity, serves as a vehicle for energy (FAs) transport, provides
ligands for nuclear hormone receptors and signal transduction processes as well as substrates for the synthesis of lipids that maintain
the permeability barrier [710]. Excessive accumulation of TG in
human adipose and non-adipose tissues, however, is related to a
variety of pathological conditions such as obesity, type 2 diabetes,
coronary heart disease, hypertriglyceridemia and non-alcoholic
fatty liver disease [1113]. With increasing prevalence of these diseases, there is considerable interest in a better understanding of TG
metabolism, which is a potential target for pharmacological intervention. In the case of plants, TG is the main component of seed
oils functioning as an energy reservoir for the young seedling after
germination until the plant develops phototrophy [14]. In addition,
TG is required for pollen and seed development [15,16]. Vegetable
oils are of great value for human nutrition, consumed primarily for
food as a source of essential FAs [17,18]. Similarly, in fungi and
some bacteria, TG serves as energy storage compound also playing
an essential role in cell growth and development [2,3,15,1922]. In
recent years, because of the raised concern of developing renewable and environmentally-benign alternatives for crude oil, TGs
produced by plants and microorganisms have been regarded as potential resources for producing biofuels and chemical feedstocks
[17,18,23]. Therefore, while limiting TG production in humans
can represent potential treatment for metabolic-related disease,
enhancing tailor-made TG accumulation in plant seeds and microorganisms is a rapidly expanding area in biotechnology [24].
1.1. TG biosynthesis
TG biosynthesis has been extensively studied since the 1950s.
The complexity of TG synthesis is reected by the existence of mul-
352
Fig. 1. Triacylglycerol (TG) biosynthesis in eukaryotes and prokaryotes [2,3,7,30,3439]. (A) Schematic representation of chemical structures of related substrates. LPA,
lysophosphatidic acid; PA, phosphatidic acid; LPC, lysophosphatidylcholine; PC, phosphatidylcholine; G3P, sn-glycerol-3-phosphate; MG, sn-1-monoacylglycerol; DG, sn-1,2diacylglycerol; TG, triacylglycerol; (B) Pathways for TG synthesis in animals, plants, fungi and bacteria. Conserved sequential acylation is enclosed in shaded area. FA, fatty
acid, GPAT, acyl-CoA:sn-glycerol-3-phosphate acyltransferase; LPAT, acyl-CoA:lysophosphatidic acid acyltransferase; PAP, phosphatidate phosphatase; DGAT, acylCoA:diacylglycerol acyltransferase; MGAT, acyl-CoA:monoacylglycerol acyltransferase; DGTA, diacylglycerol transacylase; PDAT, phospholipid:diacylglycerol acyltransferase;
WS/DGAT, wax ester synthases/acyl-CoA:diacylglycerol acyltransferase.
353
Fig. 2. Dendrogram indicating the relatedness of protein sequences displaying DGAT activities was derived from the multiple sequence alignment. The organisms and
GenBank accession numbers for proteins used are as follows: Alcanivorax borkumensis, Ab, AbWS/DGAT, YP_694462.1; Ac, Ah, Arachis hypogaea, AhDGAT3, AY875644; A.
thaliana, At, AtDGAT1, NM_127503, AtDGAT2, NP_566952; AtWS/DGAT, AtDCR, AED93236, Brassica napus, Bn, BnDGAT1, AAD45536; Caenorhabditis elegans, Ce, CeDGAT1,
CAB07399, CeDGAT2a, CAB04533, CeDGAT2b, AAB04969; Homo sapiens, Hs, HsDGAT1, NP_036211 HsDGAT2, AAK84176, HsMGAT1, AAK84178, HsMGAT2, AAI03877,
HsMGAT3, AAO63579; Mus musculus, Mm, MmDGAT1, NP_034176, MmDGAT2, AAK84175, MmMGAT1, AAI06136, MmMGAT2, AAH52831; Mycobacterium tuberculosis, Mt,
MtWS/DGAT1, NP_217646.1; MtWS/DGAT2, NP_218251.1; MtWS/DGAT3, NP_217604.1; Ricinus communis, Rc, RcDGAT1, AAR11479, RcDGAT2, AAY16324; Streptomyces
avermitilis, Sa, SaWS/DGAT, NP_828432; S. cerevisiae, Sc, ScDGAT2, NP_014888; Schizosaccharomyces pombe, Sp, SpDGAT2, XP_001713160; Umbelopsis ramanniana, Ur,
UrDGAT2a, AAK84179, UrDGAT2b, AAK84180; Vernicia fordii (tung), Vf, VfDGAT1, ABC94471, VfDGAT2, ABC94474.
genes have also been identied which encode MGATs, wax ester
(WS) synthases [66,67] and members of the BAHD2 acyltransferases which represent a large family of acyltransferases that utilize
CoA thioesters [68,69] (Fig. 2).
2.1. Dgat1
The rst DGAT gene was cloned from mouse in 1998 [64] based
on sequence homology between an expressed sequence tag and a
previously identied acyl-CoA:cholesterol acyltransferase (ACAT,
EC 2.3.1.26), a related enzyme that synthesizes cholesteryl esters
(CE) [70]. Functional expression of the mouse and human DGAT1
cDNA in insect cells showed that the encoded protein possessed
signicant DGAT activity but did not catalyze CE synthesis [64].
After this initial discovery, DGAT1 orthologs from a number of
other species, such as thale cress (Arabidopsis thaliana), were
quickly identied [7173]. DGAT1 was not found in the genome
of S. cerevisiae and most of yeast organisms, although the product
2
BAHD is named according to the rst four characterized members (BEAT or
benzylalcohol O-acetyltransferase from Clarkia breweri; AHCTs or anthocyanin Ohydroxycinnamoyltransferases from Petunia, Senecio, Gentiana, Perilla, and Lavandula;
HCBT or anthranilate N-hydroxycinnamoyl/benzoyltransferase from Dianthus caryophyllus; DAT or deacetylvindoline 4-O-acetyltransferase from Catharanthus roseus)
[69].
354
2.2. Dgat2
DGAT2 was initially puried from the lipid body fraction of the
fungus Umbelopsis ramanniana, (formerly known as Mortierella
ramanniana) [65]. The gene encoding this polypeptide did not
share any homology with DGAT1 or ACAT1/2 and thus was classied
as DGAT2. In the same study, orthologs from S. cerevisiae and
Caenorhabditis elegans were also cloned and shown to encode proteins with DGAT activity. These results formed the basis for the
identication of members from this new DGAT gene family in
mouse and human [77]. Studies using gene targeting in mice suggested that DGAT2, more so than DGAT1, plays a predominant role
in TG biosynthesis in mammals [9,78]. While DGAT1 is virtually
absent in fungi, with the exception of Y. lipolytica as previously
described, DGAT2 is widely present in nature. In S. cerevisiae and
Y. lipolytica, DGAT2 (Dga1 in S. cerevisiae and DGA2 in Y. lipolytica)
have been conrmed to make a major contribution to TG synthesis
[19,79].
Molecular characterization of DGAT2 from plants has been challenging, probably because early attempts to functionally express a
DGAT2 cDNA from model plant A. thaliana were not successful. Synthesis of A. thaliana DGAT2 in yeast was blocked at the translational level, resulting in no protein accumulation [80]. This could
be a result from differences in codon usage between the yeast
and plants [8184]. However, functional studies indicated that A.
thaliana DGAT2 gene does not encode a functional enzyme in A. thaliana either. An insertion knockout in A. thaliana DGAT2 gene did
not produce signicant reduction in seed oil content, an effect that
was not compensated for by other DGAT-related enzymes [15].
Therefore, DGAT2 does not appear to play a substantial role in
TG synthesis in developing seeds of A. thaliana.
Despite these negative results in A. thaliana, functional plant
DGAT2s were characterized from species that produce oils enriched with unusual FAs [8587], such as castor bean (Ricinus communis), tung tree (Vernicia fordii) and ironweed (Vernonia
galamensis), which produce TGs enriched in ricinoleic (12-OH
18:1cisD9), a-eleostearic (18:3 cisD9, transD11, transD13) and
vernolic (12-epoxy, cisD9 octadecenoic) acids, respectively. These
plant DGAT2s were functionally characterized and found to have
a preference for substrates containing these unusual FAs. These
studies not only conrmed that, in certain plant species, DGAT2
plays a role in the biosynthesis of TG, but also highlighted the possible function of this enzyme in selecting substrates to be incorporated into TG. Further studies with plant DGAT2, including probing
potential protein interactions, and subcellular localization, will
help determine the molecular mechanisms involved in TG synthesis by DGAT1 and DGAT2.
355
Table 1
Acyl-CoA:diacylglycerol acyltransferases (DGAT) functionally characterized in recombinant organisms. The two-character code preceding each DGAT gene indicates the organisms
of origin are as follows: Ab, Alcanivorax borkumensis; Ac, Acinetobacter calcoaceticus At, Arabidopsis thaliana; Ah, Arachis hypogaea; Bn, Brassica napus; Ce, Caenorhabditis elegans; Ea,
Euonymus alatus; Ha, Helianthus annuus; Hs, Homo sapiens; Mm, Mus musculus; Mt, Mycobacterium tuberculosis; Nt, Nicotiana tabacum; Oe, Olea europaea; Pt, Phaeodactylum
tricornutum; Rc, Ricinus communis; Sa, Streptomyces avermitilis; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Tg, Toxoplasma gondii; Tm, Tropaeolum majus; Ur,
Umbelopsis ramanniana; Vf, Vernicia fordii; Vg, Vernonia galamensis; Zm, Zea mays. Adapted from Siloto et al. [24].
Gene
cDNA
Reference
AtDGAT1
[117]
[73]
[236]
[236]
[166]
[237]
[227]
[168]
[227]
[237]
[104]
[238]
[236]
[123]
[239]
[85]
[115]
[240]
[118]
[177]
[167]
[241]
[242]
[77]
[121]
BnDGAT1
DGAT1
EaDGAT1
EpDGAT1
HaDGAT1
NtDGAT1
PtDGAT1
VgDGAT1
VfDGAT1
RcDGAT1
TgDGAT1
TmDGAT1
ZmDGAT1
CeDGAT1
HsDGAT1
MmDGAT1
CeDGAT2
HsDGAT2
MmDGAT2
DGAT2
RcDGAT2
VfDGAT2
ScDGAT2
SpDGAT2
MGAT
AWAT
WS/DGAT
Soluble
DGAT
YlDGAT2
HmMGAT2
HmMGAT3
MmMGAT1
MmMGAT2
AWAT1
AWAT2
AbWS/DGAT
AcWS/DGAT
MtWS/DGATs
SaWS/DGAT
AtWS/DGAT
AhDGAT3
AtDCR
DGAT1 or DGAT2, but with 13% identity with the bacterial WS/
DGAT (Fig. 2), was identied in developing peanut (Arachis hypogaea) cotyledons (referred to as AhDGAT3) [44]. Homologous protein sequences of AhDGAT3 were also found in other plant species
such as A. thaliana, soybean (Glycine max) and rice (Oryza sativa)
[44,105]. Previously, a cytosolic 10S multi-enzyme complex from
the oleaginous yeast Rhodotorula glutinis was also found to possess
DGAT activity [106]. Cross-reactivity was observed between AhDGAT3 and antibodies raised against a R. glutinis DGAT peptide obtained from this multi-enzyme complex [44], suggesting that
these two proteins share sequence similarities. Another soluble
[65]
[77]
[93]
[242]
[77]
[143]
[86]
[124]
[85]
[65]
[125]
[65]
[228]
[79]
[92,94]
[89,92]
[25,88,92,94]
[25]
[93]
[93]
[94]
[100]
[66]
[67]
[99]
[101]
[102]
[44]
[108]
356
Table 2
Structure of representative DGAT genes. Abbreviations for each gene indicating the organism of origin are described in Table 1.
DGAT1
DGAT2/MGAT
WS/DGAT
Soluble DGAT
Gene
ID
Chromosome
Number of exons
CeDGAT1
HsDGAT1
MmDGAT1
AtDGAT1
RcDGAT1
H19N07.4a
8694
13350
816464
8272569
5
8
15
2
N/A
2771
12321
9804
3531
7741
7
17
17
16
16
CeDGAT2a
CeDGAT2b
HsDGAT2
MmDGAT2
AtDGAT2
RcDGAT2
ScDGAT2
SpDGAT2
HsMGAT1
HsMGAT2
HsMGAT3
AbWS/DGAT
F59A1.10
W01A11.2
84649
67800
824315
8258757
854419
5802702
116255
610270
346606
4213840
5
5
11
7
3
N/A
15
3
2
11
7
N/A
1508
1385
32802
29051
2165
3450
1257
1487
38193
13398
5293
1374
2
5
8
8
8
9
1
4
6
6
7
1
SaWS/DGAT
AtWS/DGAT
AtDCR
1217410
833704
832459
N/A
5
5
1344
2751
3465
1
7
2
357
expression of DGAT2 occurs in a wider range of tissues than in humans. Modulating DGAT expression in muscle, adipose tissue and
pancreatic b-cells can affect insulin activity, adiposity and deterioration of tissue function [55].
Tissue expression of MGAT genes, which share signicant sequence similarity with mammalian DGAT2, has been studied in
mammals, as well. Much like DGAT1 and DGAT2, MGAT1 and MGAT2
are both expressed in adipose tissue as well as in other tissues
[92,93]. Notably, MGAT1 is highly expressed in stomach and kidney
and is not expressed in the intestine, while MGAT2 is predominantly expressed in the small intestine of mice/humans, but also
in the human liver. MGAT3 expression has been only detected in
the human gastrointestinal tract with the highest levels of expression occurring in the ileum [89]. Turkish et al. [93] showed that
MGAT3 is also expressed in the liver. Because MGAT transcripts
were found in other tissues in addition to intestine, where MGAT
plays a key role in dietary fat absorption, this indicates that MGAT
is involved in other aspects of lipid metabolism in other tissues in
mammals. Indeed, MGAT2 knock-out mice are protected from metabolic diseases induced by a high-fat diet, indicating MGAT2 plays
a key role in energy metabolism in response to dietary fat [28]. The
expression of MFAT/AWAT is predominant in the skin [93]. Using
in situ hybridization, Yen et al. [94] further demonstrated that MFAT/AWAT2 transcripts are restrained in sebaceous glands where
skin surface lipids (predominantly wax monoesters) are synthesized, conrming the previous nding by Cheng and Russell [114].
In plants, particularly oilseed crops, the expression pattern of
DGAT has been extensively studied, especially during seed development. Generally, expression of DGAT1 or DGAT2 is closely correlated with oil deposition in developing seeds. However, a quite
different scenario is found for the accumulation of the transcripts
Table 3
Expression pattern of DGAT genes in plants. A compilation of the results from many studies reporting plant DGAT transcript accumulation is displayed. All tissues analyzed in each
study are indicated (dap, days after pollination).
Organism
Gene
Ref.
Arabidopsis thaliana
DGAT1
DGAT1
Stems, leaves
Stems
[117]
[73]
[50]
[50]
[50]
[50]
[50]
[119]
[119]
[243]
Stems
[50]
[50]
[86]
[86]
Leaves
[50]
[118]
[239]
[50]
Roots, leaves
Roots, stems, early maturing seeds
Leaves
Leaves
[50]
[50]
[50]
[85]
[85]
DGAT1
Euphorbia lagascae
Glycine max
DGAT2
DGAT1
DGAT1
DGAT2
Stems
Young developing embryos
Young developing embryos
Olea europaea
DGAT1
Embryos, mesocarps
DGAT2
Mesocarps
DGAT1
Ricinus communis
Stokesia laevis
Tropaeolum majus
Vernonia galamensis
Vernicia fordii
DGAT1
DGAT2
DGAT1
DGAT2
DGAT1
DGAT1
DGAT1
DGAT1
DGAT2
DGAT2
DGAT2
DGAT1
DGAT2
358
Over the last few years, substantial attention has been devoted
to studying the substrate specicity of DGATs, especially those
from plants. A main motivation for conducting these studies was
to identify DGATs which could preferably incorporate desired acyl
groups into TG. A comparative study was conducted for DGAT1
versus DGAT2 from tung tree (V. fordii). Gene expression analysis
discussed in the previous section suggested that tung DGAT2, but
not DGAT1, preferentially incorporated a-eleostearic acid
[18:3cis(9,trans(11,13] into TG in seed oil bodies. Results from
in vitro enzyme assays using microsomal proteins indicated that
both DGATs have similar preferences for DG and acyl-CoA containing common FAs (palmitic [16:0], oleic [18:1cisD9], linoleic
[18:2cisD9,12], and a-linolenic acids [18:3cisD9,12,15]) [85]. Tung
DGAT2 had a lower activity than DGAT1 in vitro, which is in agreement with ndings based on enzyme assays with other plant and
mammalian orthologs of the enzymes. Through an in vivo experiment, however, it was shown that tung DGAT2 displays approximately vefold preference for production of trieleostearin than
DGAT1 by comparing the amount of TG synthesized by each isoform. Collectively, these results may suggest that DGAT catalysis
may involve other elements from the host, such as soluble cytoplasmic cofactors, absent in in vitro assays that normally utilize
microsomal membrane fractions. Discrepancies between results
from assay in vitro and in vivo studies on DGAT1 and DGAT2 have
been noticed in many studies. In rat hepatoma cells, for example,
DGAT2 overexpression led to considerably more TG accumulation
than did DGAT1 overexpression [78], although DGAT2 activity
was relatively lower in vitro [77]. It is worth noting, that in this
work, Shockey et al. [85] used an innovative approach to investigate the substrate preferences of tung DGAT in vivo. As a free FA,
a-eleostearic acid is considerably reactive and polymerizes quickly
upon exposure to air. Therefore, yeast cultures were grown in the
presence of tung oil, which contains trieleostearin, which is less
readily oxidized than the free FA. To enable the release of a-eleostearic acid from TG and subsequent import of the liberated a-eleostearic acid by yeast cells, the authors included a non-specic
lipase from Candida rugosa in the medium. By expressing tung tree
DGAT in a yeast knockout that is defective in TG synthesis, the
authors were able to determine substrate specicity for a single
DGAT. Similar strategies, based on exogenous FA supplementation
and the use of yeast knockouts, have been used by others [123].
Substrate preferences of plant DGAT2 for unusual fatty acyl
groups have also been reported for castor bean and Vernonia galamensis DGAT2 which display higher selectivity for substrates containing ricinoleic and vernolic acid, respectively [86,87,124]. When
compared with expression prole studies [50], these data provide
359
Fig. 4. Kyte-Doolittle hydropathy plots of DGAT. Hydropathy plots from representative polypeptide sequences of DGAT were generated by the method of Kyte and Doolittle
[141] using a window size of 19. Polypeptides include DGAT1 from mouse (A), oleaginous yeast Yarrowia lipolytica (YlDga2p) (B), and tung tree (C) as well as DGAT2 from
mouse (D), baking yeast Saccharomyces cerevisiae (ScDga1p) (E), and tung tree (F). The most hydrophobic segments which may contain putative TMDs are indicated by roman
numerals.
support for the hypothesis that DGAT2 may play a role in the selective accumulation of unusual FAs in the seed oil of certain plants.
Certain substrate preferences of fungal DGAT2 orthologs have also
been observed. DGAT2 from U. ramanniana showed enhanced
activity towards medium-chain fatty acyl-CoAs (e.g. 12:0-CoA)
and enhanced preference for DG containing short- and mediumchain fatty acyl groups (6:0, 8:0 and 10:0) [65]. S. cerevisiae DGAT2
used 18:1-CoA or 16:0-CoA more efciently than 14:0-CoA, 18:0CoA (18:0), arachidonyl (20:4 cisD5,8,11,14)-CoA, or linoleoyl
(18:2 cisD9,12)-CoA [74]. In Saccharomyces pombe, DGAT2 was
found to prefer palmitoyl over oleoyl moieties [125]. Recently, in
Claviceps purpurea, which produces TGs rich in ricinoleic acid,
DGAT2 was demonstrated to prefer ricinoleoyl-CoA (as an acyl donor over 18:1-, 18:2- or a-18:3-CoA [126]. C. purpurea DGAT2
could be an attractive candidate gene for engineering of oilseed
crops to produce high levels of hydroxy FAs, which will be expanded upon later in the review.
4.3. Modulators of DGAT activity
Certain proteins and electrolytes have been shown to activate or
inhibit microsomal DGAT activity in a concentration-dependent
360
Table 4
Predicted TMDs in representative DGAT. The numbers between brackets denote the bibliographical reference for each algorithm.
DGAT
DGAT1
Program
MmDGAT1
YlDGAT1 (Dga2p)
VfDGAT1
MmDGAT2
DGAT2
ScDGAT2 (Dga1p)
VfDGAT2
HMMTOP [244]
TMHMM [245]
SVMtm [246]
SOSUI [247]
TOPCONS [248]
TMpred [249]
TMDs
Orientation*
TMDs
Orientation
TMDs
Orientation
9
IN
10
IN
10
IN
8
IN
8
IN
8
IN
8
N/A
9
N/A
9
N/A
6
N/A
5
N/A
8
N/A
9
IN
9
IN
9
IN
8
IN
10
OUT
9
IN
TMDs
Orientation
TMDs
Orientation
TMDs
Orientation
1
IN
4
IN
6
IN
1
IN
1
IN
3
IN
2
N/A
3
N/A
3
N/A
2
N/A
3
N/A
2
N/A
2
IN
3
IN
2
IN
2
IN
3
IN
6
IN
361
Fig. 5. Alignment of transmembrane domains (TMDs) in DGAT1. The putative TMDs of DGAT1 polypeptides from 12 animal and 18 plant organisms were predicted and the
polypeptides were aligned. The identity of the alignment is graphed on the top using a window size of 6. The thick lines represent the sequence of each DGAT1 and the thin
lines represent the gaps generated by the alignment. The arrows denote the predicted TMDs. Note the higher level of positional conservation of the rst 4 and last 3 TMDs. The
gure was generated with Geneious Pro 5.3.4 (Biomatters) and optimized manually to t the publication format while preserving the relative positions of the TMDs and
conserved blocks. The TMDs were predicted using the Hidden Markov model (TMHMM).The organisms and GenBank accession numbers are described in the legend of Fig. 2
with the following additions: Aedes aegypti, Aa,AaDGAT1, XP_001658299; Drosophila melanogaster, Dm, DmDGAT1, AAL78365; Danio rerio, Dr, DrDGAT1, NP_956024;
Euonymus alatus, Ea, EaDGAT1, AAV31083; Glycine max, Gm, GmDGAT1, AAS78662; Jatropha curcas, Jc, JcDGAT1, ABB84383; Monodelphis domestica, Md, MdDGAT1,
XP_001371565; Medicago truncatula, Mt, MtDGAT1, ABN09107; Nicotiana tabacum, Nt, NtDGAT1, AAF19345; Nematostella vectensis, Nv, NvDGAT1, XP_001639351; Olea
europaea, Oe, OeDGAT1, AAS01606; Perilla frutescens, Pf, PfDGAT1, AAG23696; Physcomitrella patens, Pp, PpDGAT1, XP_001770929; Populus trichocarpa, Pt, PtDGAT1,
XP_002330510; Rattus norvegicus, Rn, RnDGAT1, BAC43739; Sus scrofa, Ss, SsDGAT1, NP_999216; Trichoplax adhaerens, Ta, TaDGAT1, XP_002112025; Toxoplasma gondii, Tg,
TgDGAT1, AAP94209; Tropaeolum majus, Tm, TmDGAT1, AAM03340; Vernonia galamensis, Vg, VgDGAT1, ABV21945; Vitis vinifera, Vv, VvDGAT1, CAN80418; Zea mays, Zm,
ZmDGAT1, ABV91586. Adpated from Siloto et al. [24].
Other recent work indicated that DGAT1 could have two different topologies as a result of modulation of different physiological
states [133]. Taking advantage of the differential sensitivity of
DGAT1 to specic inhibitors, Wurie et al. [133] demonstrated that
approximately equal DGAT1 activities were detected in situ in both
cytosolic (overt) and luminal (latent) sides of the ER membrane
[133]. Also, loss of both cytosolic and luminal DGAT activities were
observed in microsomal vesicles prepared from liver of DGAT1
knockout mice, indicating that DGAT1 could have a dual topology.
Wurie et al. [133] proposed that the variance between experimental evidence from in situ and in vitro data could possibly result from
the inherent limitation of the methodology used for characterizing
topology in vitro. The strategy used by McFie et al. [145] was to
probe the accessibility of a protease to heterologously overexpressed DGAT1 containing FLAG and Myc epitope tags. Although this
approach could affect the proper insertion of the protein into
membrane, it is a generally accepted and widely used method for
362
Fig. 6. Alignment of transmembrane domains (TMDs) in DGAT2 and MGAT. The putative TMDs of DGAT2 polypeptides from 13 fungus, 6 animal and 11 plant organisms as
well as 5 MGATs from animals, were predicted as described in Fig. 5 and the polypeptides were aligned. The identity of the alignment is graphed on the top using a window
size of 9. The arrows denote the predicted TMDs. Note that the rst two transmembrane domains are conserved in very similar positions based on the alignment. The thick
lines represent the sequence of each DGAT1 and the thin lines represent the gaps generated by the alignment. The gure was generated as described for Fig. 5. The organisms
and GenBank accession numbers are described in the legend of Fig. 2 with the following additions: Ajellomyces capsulatus, Ac, AcDGAT2, XP_001540241; Aspergillus oryzae, Ao,
AoDGAT2, XP_001822244; Branchiostoma oridae, Bf, BfDGAT2, XP_002208225; Bos taurus, Bt, BtDGAT2, CAD58968; Coccidioides immitis, Ci, CiDGAT2, XP_001240299;
Chlamydomonas reinhardtii, Cr, CrDGAT2, XP_001693189p; Dictyostelium discoideum, Dd, DdDGAT2, XP_635762; Laccaria bicolor, Lb, LbDGAT2, EDR14458; Medicago
truncatula, Mt, MtDGAT2, ACJ84867; Neosartorya scheri, Nf, NfDGAT2, XP_001261291; Oryza sativa, Os, OsDGAT2, NP_001057530; Ostreococcus tauri, Ot, OtDGAT2,
CAL58088; Penicillium marneffei, Pm, PmDGAT2, XP_002146410; Physcomitrella patens, Pp, PpDGAT2, XP_001777726; Populus trichocarpa, Pt, PtDGAT2, XP_002317635;
Talaromyces stipitatus, Ts, TsDGAT2, EED21737; Ustilago maydis, Um, UmDGAT2, XP_760084; Vitis vinifera, Vv, VvDGAT2, CAO68497; Zea mays, Zm, ZmDGAT2, ACG38122.
Adapted from Siloto et al. [24].
topology [133]. Increases in liver TG content, however, were observed with over-expression of DGAT1 or DGAT2, without noticeable difference in very-low-density lipoprotein (VLDL) TG levels
[147]. Luminal DGAT activities are widely considered to catalyze
the synthesis of TG for VLDL formation since the cytosolic TG droplet cannot be incorporated en bloc into VLDL [54,148,149].The phenomenon of dual topology in membrane proteins is not new [150]
363
Fig. 7. Proposed topology model of murine and yeast DGAT2 (Dga1p) [142,143]. These two models showed that MmDGAT2 has two TMDs whereas ScDGAT2 contains four
TMDs. Both termini of two DGAT orientated toward the cytosol. Important functional motifs are represented by lled blue hexagons. Black-lled residues in ScDGAT2
represent regions where topology was established based on in silico analysis only. Topology of other regions was experimentally determined.
droplets in the cytosol [54]. The proposed topology model for murine DGAT2 indicates that DGAT2 could possibly be involved in the
synthesis of cytosolic TG deposition. A recent study using stableisotope labeled substrates combined with type-specic specic
DGAT inhibitors indicated that in mice, DGAT2 preferably incorporates endogenous FAs into exogenously added glycerol [152].
DGAT1, however, displayed a preference for incorporating exogenously added FAs to endogenous DG. It is then possible, that in
hepatocytes, DGAT1 is responsible mainly for the esterication of
dietary FAs while DGAT2 acts predominantly on the esterication
of de novo generated DG through the Kennedy pathway destined
for cytosolic lipid droplets. Despite these and other lines of evidence, it is still uncertain whether the topology of DGAT1 or
DGAT2 really inuences the destination of TG (cytosol versus
lumen).
Determination of the membrane topology for S. cerevisiae
DGAT2 [142] pointed to four TMDs. As represented in Fig. 7, the
conserved functional motif YFP, and the hydrophilic segment
exclusive to yeast DGAT2, reside in a long ER luminal loop following the rst TMD. The motif HPHG is embedded in the membrane.
These results indicate some similarities to the topology model of
murine DGAT2, but also reveal striking differences. Hydropathy
analysis of DGAT2 showed divergences between animal, plant
and fungal proteins (Fig. 4, Table 4). Because considerable differences have been detected in the topology of animal versus yeast
DGAT2, it would be valuable to explore in greater detail the topological organization of plant DGAT2.
Topological characterization of S. cerevisiae DGAT2 also indicated that active and/or binding sites of each DGAT are possibly located on the luminal side (see highlighted motif in Fig. 7), which
provides evidence for an acyl-CoA-dependent TG pathway in the
lumen. This hypothesis is supported by previous work in which
luminal DG and acyl-CoA was proposed to be generated by microsomal lipase and a microsomal carnitine acyltransferase, respectively [153,154]. The existence of this mechanism has been
364
Fig. 8. Kyte-Doolittle hydropathy plots of MGATs. Hydropathy plots from representative polypeptide sequences of MGATs were generated by the method of Kyte and
Doolittle [141] using a window size of 19. Polypeptides include MGATs from humans MGAT1 (A), MGAT2 (B) and MGAT3 (C) and murine MGAT1 (D) and MGAT2 (E). The most
hydrophobic segments which may contain putative TMDs are indicated by roman numerals.
365
HsMGAT1
HsMGAT2
HsMGAT3
MmMGAT1
MmMGAT2
Program
HMMTOP [244]
TMHMM [245]
SVMtm [246]
SOSUI [247]
TOPCONS [248]
TMpred [249]
TMDs
Orientation*
TMDs
Orientation
TMDs
Orientation
4
IN
1
IN
1
IN
1
IN
1
IN
1
IN
2
N/A
2
N/A
3
N/A
2
N/A
1
N/A
1
N/A
2
IN
2
IN
2
IN
4
IN
3
IN
5
OUT
TMDs
Orientation
TMDs
Orientation
4
IN
3
IN
1
IN
1
IN
2
N/A
4
N/A
2
N/A
1
N/A
2
IN
2
IN
3
IN
3
IN
366
Fig. 9. Alignment of the hydrophilic N-terminus polypeptide sequence of DGAT1 from plants and animals. The positions corresponding to the cluster of arginines and the end
of the rst exon in plants are indicated. Accession numbers and abbreviations for the DGAT polypeptides can be found in the legend of Figs. 2 and 5.
367
Fig. 10. Alignment of some conserved sites in DGAT1 polypeptides. The bar and horizontal arrows represent the A. thaliana DGAT1 polypeptide and the predicted TMDs,
respectively. The position of the MBOAT motif is indicated above. Motifs 17 correspond to the conserved regions described by Cao [155] as follows: Motif1 (GL block), Motif2
(KSR block), Motif3 (PTR block), Motif4 (QP block), Motif5 (LWLFFEFDRFYWWNWWNPPFSHP block), Motif6 (LQL block) and Motif7 (NGQPY block). The name of each block
corresponds to the order of conserved amino acids in each block rather than a conserved sequence. The vertical boxes contain the amino acid sequences for different DGAT
indicated on the left. The vertical arrows on these boxes indicate the position of relevant amino acids discussed on the text. From left to right the conserved motifs/residues
are RXXXE, HXXXD, HXXXXD, N and H conserved in the MOBAT family. Accession numbers and abbreviations for the DGAT polypeptides can be found in the legend of Figs. 2
and 5.
368
the same motif is largely conserved and precedes the third last
hydrophobic segment. The variant FYQQWWN is found in DGAT1
from Y. lipolytica. In this motif, substitution of the conserved tyrosine in DGAT1 from T. majus (Y392A) resulted in decreased enzyme
activity while a double mutation in tyrosine and tryptophan
(Y392G/W395G) completely abolished enzyme activity [118].
Polymorphisms analysis in DGAT1 in natural populations has
also provided valuable information on the structure/function relationships of these enzymes. It is also helpful for conrming the role
of DGAT1 for TG biosynthesis in vivo in the original organisms. This
is the case of a non-conservative substitution of lysine to alanine in
DGAT1 from cattle, which is a quantitative trait locus (QTL) for
milk fat [176]. This lysine residue is conserved in most animals,
but not in plants. Another DGAT1 QTL was demonstrated for corn,
where the presence of a phenylalanine residue in position 469 of
corn DGAT1 increased the enzyme activity and overall seed oil content in corn [177]. This phenylalanine residue is conserved in most
DGAT1s from plants.
6.2. Conserved and functional motifs in DGAT2
DGAT2 polypeptides have fewer hydrophobic regions than
DGAT1. A hydrophobic region that might represent one or two
Fig. 11. Alignment of some conserved sites in DGAT2 polypeptides. The bar and horizontal arrows represent the U. ramanniana DGAT2a polypeptide and the predicted
transmembrane domains, respectively. Motifs 1 to 7 correspond to the conserved regions described by Cao [155] as follows: Motif1 (PH block), Motif2 (PR block), Motif3 (GGE
block), Motif4 (RGFA block), Motif5 (VPFG block), Motif6 (G block). The name of each block corresponds to the order of conserved amino acids in each block rather than a
conserved sequence. The vertical boxes contain the amino acid sequences for different DGAT indicated on the left. From left to right the conserved motifs/residues are YFP, (H/
E)PH(G/S), GGXXE, and RXGFX(K/R)XAXXXGXX(L/V)VPXXXFG(E/Q). Accession numbers and abbreviations for the DGAT polypeptides can be found in the legend of Figs. 2 and
6.
adjacent hydrophobic domains is conserved in the N-terminal portion (Fig. 6). The hydrophilic N-terminus preceding the rst hydrophobic segment is variable in length, and is much larger in
polypeptides from fungi than those from animals and plants [24].
A comparison of 54 DGAT2 polypeptides indicated that these proteins are composed of an average of 344 amino acid residues [155].
DGAT2 polypeptide sequences across species were more diverse
than those for DGAT1. Cao [155] reported 4.7% of identity of protein sequences while we detected only 2.3% [24]. From an experimental perspective as discussed earlier, the higher divergence in
DGAT2 genes represents a hurdle for the identication of the ensuing cDNAs from organisms whose genomes that have not yet been
sequenced.
As demonstrated in Fig. 10, seven regions with remarkable sequence conservation have been located by Cao [155]. The most
conserved region in DGAT2 corresponds to the motif RXGFX(K/
R)XAXXXGXX(L/V)VPXXXFG(E/Q) located approximately 150 residues after the rst hydrophobic segment (Fig. 11). As described
in previous section, a cysteine 314 resides in close proximity to this
motif in S. cerevisiae DGAT2. Although replacement of this cysteine
to alanine did not affect enzyme activity, thiol-specic modication on this cysteine substantially inhibited ScDGAT2 activity.
These data indicate that attachment of thiol-specic group to cysteine 314 may interfere with the interaction of key amino acids in
this molecular signature, which eventually resulted in the loss of
enzyme activity [132]. Therefore, the motif mentioned above could
contribute to an important site for DGAT catalysis. Other conserved
regions include the motif GGXXE (motif 3 in Fig. 11) and a phenylalanine, arginine and proline in positions 164, 170 and 293, respectively, of U. ramanniana DGAT2a. In addition, the motif HPHG
(EPHS/G in plants) is conserved in sequences from animals and
fungi (Fig. 11). Preceding this motif, the most evident structural
divergence in DGAT2 is observed; a hydrophilic segment of
approximately 38 residues present in sequences from some fungi,
but absent in plants and animals. Though not essential for the enzyme activity, this region inuences the activity of S. cerevisiae
DGAT2 (positions 150187) [142], and might represent a specialized domain in this enzyme. These conserved motifs discussed
above have also been identied by Cao [155] using a large number
of DGAT2 polypeptides (Fig. 11).
The importance of several conserved regions in DGAT2 has been
determined. Stone et al. [143] demonstrated that the motif HPHG
conserved in both animal and fungi DGAT2 (Fig. 11) could be part
of the active site as it was essential for the activity of murine
DGAT2. Site-directed mutagenesis indicated that the second histidine residue in this motif appeared to play a predominant role in
enzyme function. Similarly, in S. cerevisiae, replacing the HPHG sequence by EPHS, conserved in plant orthologs, was detrimental to
enzymatic activity [142]. In addition, substitution of the second
histidine to alanine abolished enzyme activity. These results suggest that HPHG, and more specically the second histidine residue,
are essential for enzyme catalysis. Also, this motif could serve as a
candidate to explore possible important structural and functional
divergence between DGAT2 from animals, fungi and plants. Substitutions on another conserved motif YFP, located close to the TMDs
(Fig. 11), resulted in signicant decreases in activity in S. cerevisiae
DGAT2 [142].
The motif FLXLXXXn (n = non-polar residue) was proposed to
represent a neutral lipid binding domain of murine DGAT2 [143].
This motif was identied in several enzymes that bind to or catalytically act on neutral lipids, including TG hydrolase, hormonesensitive lipase, cholesterol ester transfer protein, and cholesterol
esterase [143,178,178]. Amino acid substitutions on the phenylalanine and rst leucine in this motif decreased DGAT activity, while
substitution of the second leucine abolished activity. This motif, located in the rst TMD of murine DGAT2, is present in most of
369
370
Fig. 12. Multiple sequence alignment showing conserved residues in the C-termini of DGAT. DGAT1 (A) and DGAT2 (B) polypeptides were aligned using Geneious (Biomatters
Ltd.). Accession numbers and abbreviations for the DGAT polypeptides can be found in the legend of Figs. 2, 5 and 6. The bars on top indicate the positions of the identied ER
retrieval motifs for tung tree DGAT1 (YYHDL) and tung tree DGAT2 (LKLEI).
371
372
Table 6
Genetic engineering of oilseed plants through the use of DGAT [36]. Reprinted with permission of Elsevier Science Ltd.
Host species
Source species
Up-regulated gene
Phenotype
Ref.
B. napus
A. thaliana, N. tabacum
Glycine max
A. thaliana
B. napus, A. thaliana
A. thaliana
U. ramanniana
R. communis
[227,230]
[166,236]
[228]
[124]
A. thaliana, B. napus
Glycine max
T. majus
V. galamensis
DGAT1
DGAT1
DGAT2
FAH12
DGAT2
DGAT1
DGAT1
DGAT2
EPX
[118]
[231]
9. Closing comments
Advances in molecular biology, biochemistry and biotechnological applications of DGAT-related proteins have facilitated a substantial increase in our understanding of the low-resolution
functional and structural relationships of these essential enzymes
involved in TG metabolism. While many enzymes possessing DGAT
activity were identied during the last fteen years, DGAT1 and
DGAT2 are the two major membrane-bound DGATs widely distributed in different organisms. As two unrelated polypeptides, catalyzing the same reaction, the simple picture of DGAT1 and
DGAT2 has recently been replaced by the more complex scenario
in which each enzyme seems to have unique functional roles
across species. Although the considerable advances have been
achieved in comparative analyses of these two enzymes, many
373
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